Our long-term goal is to understand the biochemical cause for primary open-angle glaucoma, in order to provide a cure and/or a prevention for this disease. Increased resistance to aqueous outflow to be the causal factor, and the trabecular meshwork has been implicated as the major site of this resistance. The trabecular extracellular matrix (ECM) is thought to provide the physical "barrier" to fluid movement in this region. While very little is known about the trabecular ECM, the highly-charged glycosaminoglycans (GAGs), which are an integral part of trabecular proteoglycans, are likely to play a major role in providing resistance to aqueous outflow. The immediate objective of this proposal is an understanding of the structure and interactions of the trabecular proteoglycans, of their biosynthesis and turnover and of how these activities are regulated at the second messenger level. To obtain large amounts of trabecular ECM components and to develop molecular probes to them, viral transformation of calf and human trabecular cells has been initiated, and these transformants will be characterized in detail. Both a standard transforming virus, SV40, and a recombinant hybrid virus, MV40, have been used in this effort. The latter has a very high rate of permanent transformation, and has been modified for better human infectivity. The sensitive probes that will be developed and used in these and future studies include: 1) antibodies to synthetic peptides which have been designed using the known sequences of several proteoglycans and matrix metalloproteinases (which are the likely initiators of ECM turnover); 2) synthetic oligonucleotide probes to the mRNAs of proteoglycan core proteins, matrix metalloproteinases and tissue inhibitors of metalloproteinases ("TIMPs:, which terminate metalloproteinase action); and 3) cDNA probes to these same mRNAs. The use of these probes will be coupled with basic biochemical methods that were developed in the last grant period. These probe and methods will be used to obtain information about the structures, genes, interactions, biosynthesis, turnover and regulation of the trabecular proteoglycans. The emphasis of the regulation studies will be upon the role(s) of known second messengers and related cellular modulators in the biosynthesis and turnover of the trabecular proteoglycans. This information will aid in the development of an animal model for glaucoma that can be used to test the role(s) of these ECM components in the regulation of aqueous humor outflow in the normal and pathological trabecular meshwork.